A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans

Thapakorn Jaroentomeechai, Yong Hyun Kwon, Yiwen Liu, Olivia Young, Ruchika Bhawal, Joshua D. Wilson, Mingji Li, Digantkumar G. Chapla, Kelley W. Moremen, Michael C. Jewett, Dario Mizrachi, Matthew P. DeLisa*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Original languageEnglish (US)
Article number6325
JournalNature communications
Volume13
Issue number1
DOIs
StatePublished - Dec 2022

ASJC Scopus subject areas

  • General Physics and Astronomy
  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology

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